THE IDENTIFICATION OF GENES AND ENZYMES IN THE ALDOXIME-NITRILE METABOLISING PATHWAY

Dooley-Cullinane, Tríona-Marie and Coffey, Lee V. and O'Reilly, Catherine (2019) THE IDENTIFICATION OF GENES AND ENZYMES IN THE ALDOXIME-NITRILE METABOLISING PATHWAY. PhD thesis, Waterford Institute of Technology.

Text Tríona Dooley-Cullinane 2019.pdf Download (4MB)

Abstract

Biocatalysis is gaining much attention for the production of pharmaceutical intermediates, drug compounds and, fine chemicals. Biocatalysis, as a route of synthesis, boasts many benefits over traditional chemical synthesis. It can account for enantiopure compounds, lower waste production, decreases the need for potential purification and down-stream processing while also allowing the reactions to be carried out at milder conditions, such as lower temperatures and a relatively neutral pH (Gong et al. 2012, Singh et al. 2006). Due to the often low efficacy of racemic drugs or in fact the side effects caused by one of the enantiomers, enantiopure products are highly sought after (Albarrán-Velo et al. 2018). Thalidomide, a drug that was previously prescribed to treat morning sickness during pregnancy was sold as a safe and effective anti-emetic, in its racemic form. Unfortunately, years later the level of incidents of miscarriage and foetal birth defects observed amongst the women who were prescribed the drug led to the discovery that the S-enantiomer of the drug was teratogenic. Enzymes comprising the Aldoxime-Nitrile pathway are the focus of the body of work presented in this thesis. In 2010, it was reported that there were over 30 nitrile-containing pharmaceuticals on the market with another 20 at clinical trial stage (Fleming et al. 2010). Many nitrile containing drugs or intermediates may have been produced via chemical synthesis coupled with biocatalysis. Focusing on the identification of aldoxime dehydratase enzymes can offer a new, improved route to nitrile synthesis. The research presented in chapter two and published by (Dooley-Cullinane et al. 2016) presents a new high-throughput real-time (qPCR) for the detection of novel aldoxime dehydratase genes. The potential application of these genes/enzymes was reinforced by the Food and Drug Administration (FDA) approval of 11 nitrile containing pharmaceuticals since 2010. This is an indication as to what is to come with regard to an increase in market size for bio-pharmaceuticals produced by biocatalysis, in particular nitrile containing pharmaceuticals. The remaining enzymes which comprise the aldoxime-nitrile pathway, the nitrile-degrading enzymes such as nitrilases and nitrile hydratases (NHases), have been shown to display a broad substrate scope and complex nitrile substrates can be converted into their corresponding amides with the final enzyme, and amidase converting the amide to the corresponding carboxylic acids. The area of nitrile metabolism has been of great interest and will be into the foreseeable future (Huisman and Collier 2013, Gong et al. 2012). The body of work presented, details outputs and discoveries which form a part of the larger web of research undertaken by the molecular biocatalysis research group. The overall aim of research carried out by the group is the identification and discovery of novel genes and enzymes towards the production of industrially or biopharmaceutical relevant products. This thesis presents two novel molecular methods for the detection of genes within the aldoxime-nitrile pathway. A novel qPCR method was designed for the detection of aldoxime dehydratase genes. The second molecular method presented is a novel clade-specific touchdown conventional PCR which allows for the clade specific detection of novel nitrilase genes. Additionally, this protocol allows for the detection of nitrilase genes with predicted enzymatic activity in model substrates based on their clade classification. A novel nitrilase previously identified from Burkholderia sp. LC8 was cloned, expressed and characterised with results displaying a broad substrate range and excellent enantioselectivity towards mandelonitrile. The research also documents the discovery of a novel nitrilase partial sequence through the construction of a genomic fosmid library from genomic DNA of Rhodococcus erythropolis SET-1. This body of work involved the screening of fosmid clones for functional activity on 3-HGN. Further work presented a discussion on the attempts to elucidate the full nitrilase gene sequence via fosmid insert sequencing and also whole genome sequencing which led to the identification of a nitrile hydratase/amidase genomic cluster in R. erythropolis SET-1.

Item Type:	Thesis (PhD)
Additional Information:	This was for the final master project This is a placeholder note
Departments or Groups:
Depositing User:	Derek Langford
Date Deposited:	24 Sep 2019 10:50
Last Modified:	19 Apr 2024 23:03
URI:	https://repository.wit.ie/id/eprint/3374

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